Oligonucleotide treatment of ras-induced tumors in nude mice.

Abstract

Oligonucleotides have shown an ability to target specific oncogene transcripts and inhibit their expression in cells, but the degree to which sustained treatment can suppress the levels of an oncogenic protein enough to benefit a patient remains to be determined. This question has been studied in several ways. First, the relationship of antisense DNA inhibition to the predicted secondary structure of human H-RAS oncogene mRNA was examined in transformed mouse cells that form solid tumors. Inhibition of H-Ras expression was sequence-specific, dose-dependent, and correlated with inhibition of focus formation. The efficacy of the first intron antisense sequence in reducing H-Ras expression was greater than that of the initiation codon target. Second, H-RAS transformed solid tumor cells were pretreated in vitro with normal oligonucleotides, after which tumor growth from the treated cells was tested in nude mice. The three days of treatment with the first intron antisense DNA reduced H-Ras cellular levels by more than 90% whereas a nonspecific control DNA reduced H-Ras levels by approx 20%. Tumor growth of cells treated with H-RAS antisense oligonucleotide was significantly reduced for up to 14 d following the end of treatment and implantation into the mice, whereas the nonspecific control DNA had no significant effect. Third, H-RAS transformed bladder cancer cells were implanted into nude mice, after which the mice were treated for 31 d with oligonucleotide phosphorothioates. Tumor growth in mice treated with H-RAS 12th codon antisense oligonucleotide was reduced by about 80% throughout the treatment period, reiterating the sustained effect seen in pretreated tumor cells. However, the scrambled phosphorothioate control inhibited tumor growth by about 60%, illustrating some nonspecific inhibition. Fourth, K-RAS transformed pancreatic cancer cells were treated in culture and in nude mice. Inhibition of K-Ras expression with a phosphorothioate oligonucleotide directed against a 5'-UTR sequence was sequence-specific and dose-dependent. K-RAS transformed pancreatic cancer cells were implanted into nude mice, after which the mice were treated for 14 d with oligonucleotide phosphorothioates. Tumor growth in mice treated with K-RAS 5'-UTR antisense oligonucleotide was reduced by about 50% throughout the treatment period, reiterating the sustained effect seen with H-RAS transformed cells. In this case, the sense phosphorothioate control did not inhibit tumor growth, demonstrating that nonspecific inhibition is not a characteristic of all phosphorothioate sequences. The next logical steps include testing oligonucleotide efficacy against other tumor types, toxicological testing in higher species, and clinical trials in human subjects.